Critical Periods for Prenatal Alcohol Exposure: Evidence From Animal and Human Studies.

Birth defects associated with FAS can vary, depending on when during gestation the fetus is exposed and how long the exposure continues. Although defects such as craniofacial abnormalities have been clearly associated with exposure early in pregnancy, behavioral deficits have not been as closely connected with a critical period of pregnancy.

A lthough in 1968, Lemoine and drinking during pregnancy; the duration was considered the risky period-when colleagues (1968) informed the of the fetus' exposure; and whether, if exposure might lead to birth defectsscientific and medical commu the fetus is exposed early in gestation, it whereas exposure during the later part of nity of alcohol's potential as a makes any difference to the outcome if pregnancy was not considered dangerous.teratogen (i.e., alcohol is a substance that the mother discontinues her drinking for A possible explanation for this view can affect a developing fetus, resulting in the duration of her pregnancy.These involves the kinds of defects that generally physical and neurological damage), Jones questions concern the fetus' vulnerability appear if exposure to a teratogen occurs and Smith's (1973) classic article first during different critical periods of gesta early in pregnancy.The first trimester is employed the term "fetal alcohol syn tion.This article reviews research find the critical period of organogenesis, when drome" (FAS).It is evident from this and ings from studies of humans and animals the major organs form.Exposure during later reports that maternal alcoholism and about the effects of alcohol during critical this time, and particularly during the first 2 heavy drinking during pregnancy are periods in pregnancy.months-the embryonic period-can result associated with readily observable physi in dysmorphia.The effects of teratogenic cal defects, called dysmorphia; reduced exposure during the second and third growth; and damage to the fetus' nervous

CRITICAL PERIODS
trimesters, which include growth retar system. 1 However, the extent and pattern dation and neurological defects (Scialli of alcohol exposure necessary to produce In the first half of this century, the placenta 1992), are not as obvious and may not be any of these negative effects are still was considered a natural barrier capable of under investigation.Some of the most vexing questions protecting the developing child from expo CLAIRE COLES, PH.D., is director of Clini sure to harmful agents, including disease about alcohol's effects on the fetus that cal and Developmental Research at the and toxic chemicals.When researchers remain concern the timing of a mother's Human and Behavior Genetics Labora discovered, during the 1950's and 1960's, tory of the Department of Psychiatry and that certain diseases, such as rubella, and is director of Psychological Services at attributed to exposure unless observers are aware of the potential for their occurrence.
There is another, earlier stage-pre implantation-before the fertilized egg attaches, which used to be considered a safe period because the developing em bryo is not yet connected to the maternal bloodstream and, therefore, does not appear to be receiving the teratogenic substance.In the 1980's, however, re search revealed that these ideas may not be completely accurate (Fabro et al. 1984).Many chemicals and organisms are able to cross the placental barrier.These terato gens also have been shown in some studies to affect the fetus during the earliest part of pregnancy that has been studied.
Inherent in the idea of a critical period of pregnancy is that the embryo or fetus is vulnerable during specific times when it is undergoing particular developmental processes (figure 1).This periodsensitive vulnerability can be pinpointed when a specific process in development is affected only during these windows of time.For instance, female fetuses may have genital malformations if exposed to androgens (male sex hormones) only around the 10th week of gestation, when the sexual organs are differentiating.Mercury, which has its greatest impact on the developing nervous system, affects the fetus during the second trimester, when nerve cells are prolifer ating.Other teratogens such as lead or alcohol seem to have negative effects throughout gestation, causing physical anomalies, retarded fetal growth, and neurological damage.

ANIMAL STUDIES
Several difficulties arise in attempts to study many of the effects of alcohol expo sure on humans during pregnancy.For example, investigators cannot regulate the timing and extent of the subjects' exposure to alcohol or the strength or purity of the dose, and they cannot control individual differences in response to the alcohol.In contrast, these factors can be controlled in animal studies, where investigators can regulate the dose of alcohol and monitor blood alcohol levels to assure that the exposure is consistent.In addition, to study the timing of effects, animals can be ex posed to alcohol throughout gestation, during particular periods of gestation, or only on specific days.The many social and environmental factors that limit confidence in conclusions drawn from human research also can be eliminated through animal research.For instance, the effects of nutritional deficien cies, which are often associated with heavy alcohol use, can be regulated in animals through feeding a control animal the same diet that the alcoholized animal eats volun tarily.Although this technique may not entirely control the problems of nutritional absorption, it does assure an equal intake of nutrients by both animals.
However, there are some problems in using animals as models for the effects of alcohol on humans.First, particular species of animals do not always respond to a substance in the same way humans do, and drugs are often metabolized dif ferently in different species.Second, the gestational process itself may not be com pletely analogous in different species.For example, when using a rat model to study the behavioral effects of exposure during the brain growth spurt, it is important to realize that this period, which occurs in the human third trimester, occurs after birth in the rat (figure 2).Therefore, alco hol has to be administered to rats in the period right after birth to produce the same results (West and Goodlett 1990).
Finally, the type of effects being in vestigated may limit the usefulness of animal models.For instance, researchers are concerned with studying the impact of fetal alcohol exposure on affected children's social behavior and higher cognitive functioning.However, these processes cannot be modeled satisfactori ly in most nonhuman species, which do not show deficits in aspects of behavior such as social judgment, language devel opment, or math skills.

INSIGHT FROM ANIMAL STUDIES
Despite inherent limitations, investigations of timing of alcohol exposure in animal models have led to many important insights into physical anomalies in humans.Sulik and Johnston (1983) have investigated the impact of alcohol exposure in mice and established that exposure around day 7 of gestation produces facial dysmorphia and correlated brain alterations equivalent to that seen in children with FAS.Clarren and colleagues (1988) studied effects in pri mates by exposing two groups of pigtail macaques to a range of doses of alcohol.One group was exposed throughout preg nancy, and the other was exposed after the In the rat (scale is measured in days), the brain's growth peaks after birth, whereas in humans (scale is measured in months), the brain's growth peaks at birth.Growth is measured as a percentage of adult weight.As brain growth slows, continued growth in the rest of the body causes the brain's weight to become a smaller percentage of total adult weight.fifth week of gestation (a time approxi mately equivalent to late in the first tri mester in humans).This study suggested that facial malformations occurred between gestational days 20 and 32 in this species and that early exposure was much more damaging for growth and behavior than exposure later in pregnancy, even if a larger dose was given at a later time.The re searchers also saw effects on behavior even in the absence of obvious physical defects.
From animal studies, it is clear that early exposure (first trimester) produces significant dysmorphia and neurological damage; however, there also are effects on the nervous system as a result of later exposure only.Using a rat model, Miller (1992) has investigated the effects of exposure during the second half of gesta tion (equivalent to the second trimester in humans).He notes that brain weight reduc tion is a consistent finding in animals alcoholized both in this period and in the early postnatal period (equivalent to the third trimester in humans).During the second half of gestation, nerve cells in the neocortex are generated and migrate to the appropriate brain regions.Alcohol expo sure appears to affect the timing and pat tern of nerve cell generation, both delaying the process and altering the number of cells that are produced.In addition, cell migra tion patterns are altered so that unusual cell formations can be observed in many areas in the brain, including the hippocampus,2 cerebellum, sensory nucleus, and neocor tex (Miller 1992).
Third trimester (equivalent) effects have been studied extensively in a rat model by West and Goodlett (1990).This period is of interest because it includes the brain growth spurt, a time of very rapid brain development that occurs in part during the third trimester in humans and postnatally in rats (figure 2).Exposure to alcohol during this period leads to re ductions in brain weight and head cir cumference, presumably associated with alterations in brain structure and function.The number of cells in certain regions of the hippocampus and the cerebellum are reduced.The hippocampus is known to affect learning and memory and the cere bellum to affect motor ability.That these anatomical changes are related to abnormal behavior later in life is suggested by find ings that hyperactivity and learning deficits can be observed in physically normal animals exposed to alcohol during this period (West and Goodlett 1990).

HUMAN STUDIES
Although dysmorphia, growth, and certain kinds of behavior can be studied in animal models, it is only through human clinical and epidemiologic studies that other im portant behaviors such as language devel opment can be investigated.However, researchers have to be aware of the limita tions of these studies, such as those that arise from the complex pattern and duration of alcohol use usually seen in pregnancy, which is discussed below.To investigate how timing affects outcome and to use statistical techniques most effectively, it would be best if information was available about alcohol exposure at different points during gestation; some fetuses would be exposed in the first trimester, others in the second, and still others in the third.It would be helpful also if levels or doses of drinking were the same so that only the timing of exposure was different rather than timing and amount both.
However, women who drink during pregnancy are not motivated by scientific rigor.Instead of adopting drinking pat terns that range across trimesters, they are more likely to drink heavily around the time of conception, before realizing that they are pregnant, and to reduce or stop drinking later in pregnancy.Some women reduce drinking because they are aware of the potential problems; others reduce or eliminate alcohol use because of an aver sion to its taste or because of the nausea that they feel at this time.Virtually no women begin drinking heavily only dur ing the second or third trimesters or both.Thus, it is impossible to replicate animal results in human clinical studies.Because of these patterns of use and the likelihood that the same women will drink heavily and throughout pregnancy, there are technical difficulties in using statistics to

G L O S S A R Y
Cerebellum: The second largest portion of the brain.It is involved in motor functions such as mainte nance of posture, coordination, and balance and also may be connected with emotional development.
Correlational studies: Studies of humans that relate one behavior or interaction with the environment to observed physiological or psycho logical effects.Because no study of humans can eliminate other possi ble causes of such effects (some thing that can be done in controlled animal studies), only correlations can be drawn between a behavior or teratogen and an observed effect.
Hippocampus: A component of the limbic system within the brain that is involved in emotional behaviors related to survival, such as flight or fight responses.The hippocampus is associated with memory, particularly with the learning of new informa tion and of sequentially presented information.

Neocortex:
The most recently evolved portion of the cerebral cortex.The cerebral cortex (including the neo cortex) covers the surface of the cerebrum (the largest portion of the brain) and is responsible for higher mental functions, general move ment, perception, behavioral reac tions, and the integration of these functions.

Sensory nucleus:
The nucleus of termination of the sensory fibers of a peripheral nerve.The peripheral nerves carry information from the sense organs (eyes, ears, skin) to the brain.These neurons terminate in clusters of cells called nuclei.
Information is then transferred from these sensory nuclei to other brain structures.
control for all of the effects associated with trimester of exposure.
Another difficulty involves the admin istration of alcohol.Because biological measures of alcohol use such as blood and urine tests are less accurate during preg nancy due to the presence of hormones of pregnancy, it is necessary to rely on a woman's selfreport for the amount and frequency of her drinking.Even when a woman is reporting her drinking as accu rately as she can, such reports may be in error, particularly when they involve recall over long periods of time.Because estimates of exposure, particularly expo sure during specific periods of gestation, are tentative, human research has focused not on specific days or weeks but on four broad time periods-periconception, first trimester, second trimester, and third trimester-and has asked women to esti mate, usually retrospectively, their drink ing during these periods.To get a feel for how accurate this information is likely to be, readers are encouraged to try to re member exactly how much they drank during a particular week 6 months ago.Typically, only abstainers and alcoholics are able to do this with any accuracy.
In contrast to most investigators, who ask women about their alcohol use only on one occasion (usually at the time they are recruited into the study), a few inves tigators have assessed drinking repeatedly and systematically.Day and colleagues (1989) identified women who used alco hol early in pregnancy and repeatedly interviewed them, asking about use dur ing each trimester as well as after the birth of the baby.This method allows relatively accurate estimates of exposure by trimester and allows for the investiga tion of trimester effects.
Another way to examine the issue of timing of exposure is to take advantage of an experiment that results sponta neously when pregnant women are re cruited into programs designed to help them stop drinking.Coles and colleagues (1985) and Larsson and colleagues (1985) have compared physical and behavioral differences in offspring of those women who stopped drinking as a result of inter vention, with the offspring of those who did not stop drinking in the second tri mester, to estimate the benefits of third trimester abstinence.However, it is nec essary to interpret results of such studies cautiously, because it is likely that wom en who continue to drink despite educa tional and therapeutic interventions also differ in other ways from women who are able to stop.For instance, those wom en who continue to drink also are more likely to smoke cigarettes and might be more physically addicted to alcohol than those who were able to stop.Thus, third trimester effects in the offspring of these women could stem from these differ ences and not only from consumption of alcohol (for more information, see Smith et al. 1986).

Nonviability
It is probable that heavy, extremely early alcohol exposure often leads to nonviabil ity of the fetus and spontaneous abortion.This is difficult to measure, however, because pregnancy may not have been identified and pregnancy loss may not be recognized.

Physical Anomalies (Birth Defects)
Because facial dysmorphia occurs during the embryonic period (the first 8 weeks of the first trimester), craniofacial anomalies in human subjects are probably associated with drinking during this initial stage of pregnancy.Clinical support for this period of vulnerability can be inferred from stud ies that examine the effects of stopping drinking during the second trimester (Coles et al. 1985), in which equivalent physical dysmorphia scores were noted in two separate groups.The first group included children whose mothers drank throughout gestation.The second group included children whose mothers stopped drinking in the second and third trimesters but drank amounts during the first trimester that were equivalent to those consumed during the first trimester by the mothers of the first group (mean amount reported for both groups was 24 drinks a week, with a range of 2 to 150 drinks a week).
In statistical studies of craniofacial anomalies in children exposed to alcohol prenatally, Ernhart and colleagues (1987) report that a relationship between these anomalies and first trimester exposure was evident.The anomalies also were related to later intellectual development in that greater dysmorphia was associated with lower IQ's.In their longitudinal studies, Graham and colleagues (1988) found minor physical anomalies in alcohol exposed children, present at birth and also at age 4 years, that were related to heavy drinking in the periconceptual period rather than at midpregnancy.Day and colleagues (1989) found that physical anomalies observed in infants were associ ated with reports of heavy drinking (de fined in their study as at least one drink a day, or about 0.5 ounce of alcohol) in the first 2 months of pregnancy only.

Effects on Growth
Growth is usually measured by birth weight (or weight in older children), head  circumference, and length (height).In contrast to the relationship observed with facial dysmorphia, effects on growth ap pear to be related to exposure later in pregnancy.This relationship is shown in figure 3, which presents data from studies of the effect of discontinuing alcohol and other drug use by the second trimester of pregnancy on neonatal measures of growth.Drugs other than alcohol are mentioned because it cannot be proved that cigarettes and drugs such as marijua na were not used by at least some of the women in the study. 3 These data suggest that exposure that continues throughout pregnancy produces fetal growth deficiencies that can be ob served at birth.When alcohol and other drug use is discontinued by the beginning of the second trimester, children of drink ers may approach the growth of children of nondrinkers (Coles et al. 1985;Rosett et al. 1980).These findings may indicate either that the growth deficit associated with alcohol occurs in the third trimester when the fetus is known to be growing rapidly or that being alcoholfree during this time allows the previously exposed fetus to catch up on growth.
When alcoholexposed children are studied over time, the negative effects of alcohol exposure on some aspects of growth seem to be mitigated, whereas other aspects of growth are still affected.Figure 4 shows the weight, height, and head circumferences of children in a longitudinal study, some of whom had a diagnosis of FAS or FAE (fetal alcohol effects), and who were reevaluated at early school age (5 to 7 years old).Notice that at this stage, although weight and height are no longer significantly lower in the alcoholexposed groups, head circumfer ence remains smaller among children who were exposed throughout pregnancy.These data are consistent with the results of animal studies, which found deficits in head circumference in animals exposed during the brain growth spurt (West and Goodlett 1990).
Following a larger sample (650 wom en) than that shown in figure 4, Day and colleagues (1991) also found that as chil dren aged, there were observable differ ences in growth rate that appeared to link exposure to alcohol during the second and third trimesters with the effect on height and weight as well as on head circumfer ence.In addition, due to differences in 3 The effects of cigarettes and drug use cannot be ruled out as factors in the data presented in figures 3, 4, and 5. methodology from those used in the study represented in figure 4, these investigators have identified an adverse effect on growth due to first trimester alcohol exposure, and, based on these data, they suggest that there may be more than one mechanism for alcohol's effects on growth.

Behavioral Effects
These effects have not been examined as closely in relation to critical periods of exposure as have facial features and growth, primarily because it is much more difficult to make such a connection.Studies of facial dysmorphia have pro duced a clear connection with alcohol exposure during the first trimester.How ever, because the relationship between specific brain sites and functions and most human and animal behaviors have not been identified (or may not exist), the same behavior may have more than one "cause."For instance, a child's poor atten tion may result from prenatal or traumatic brain damage, from environmental factors, or from anxiety.Therefore, observation of attention problems in alcoholexposed children does not point to a particular period of exposure in the same way that facial dysmorphia does.
In addition, children are exposed to different amounts of alcohol at different times during their gestation, so there is a wide range of possible outcomes resulting from fetal alcohol exposure.For example, the severe central nervous system damage associated with reduced head size, or microcephaly, in some FAS children probably results from different maternal drinking patterns from those that cause the mild effects, such as lower IQ scores and alterations in behavior, found in otherwise normal children.
Despite these problems in drawing conclusions from observed behavioral effects, it is possible to tease out some relationships between particular behav ioral outcomes and alcohol exposure during different periods of pregnancy.Early, heavy exposure leads to the most severe outcomes and is associated with mental retardation, sensory deficits, and motor problems.More subtle behavioral effects, such as learning disabilities and attention problems, can result from less extensive exposure.For instance, in a prospective sample (mean alcohol use by mothers before pregnancy recognition was approximately 1 drink per day, with a range of 0 to 50 drinks), Streissguth and colleagues (1989) found that reported moderate drinking (mean stated above) either before pregnancy recognition or at midpregnancy was associated with rela tively mild later deficits on neuropsycho logical tests (children scored an average of four points lower than normal on IQ tests).Early, heavier drinking (the upper limit of the range stated above) was found to result in more serious outcomes on the neuropsychological tests even in chil dren without physical effects.In seeming contrast, Larssen and colleagues (1985) found that preschool children who were exposed to alcohol throughout gestation (with a range of two to nine drinks per day) were more likely to show hyperactiv ity, language problems, and motor deficits in comparison with those whose mothers stopped drinking by the second trimester, which implies that these effects result from later exposure.
Similar results to Larssen and col leagues' were obtained in a followup of school age children (Coles et al. 1991) that focused on cognition, attention, and behav ior (figure 5).In these data, alcohol expo sure during any part of pregnancy appears to be associated with poorer academic achievement.Exposure during the third trimester in particular appears to be associ ated with lower aptitude scores, although the majority of these children cannot be Critical Periods for Prenatal Alcohol Exposure classified as mentally retarded.Effects on aptitude that are associated with third trimester exposure are probably the cumu lative effect of alcohol exposure through out pregnancy.Some of the other deficits that were seen in these children exposed through the third trimester (e.g., poorer attention and sequencing and motor prob lems) are consistent with those seen in people with damage, such as trauma, to the hippocampus and the cerebellum (Mirsky 1987).These are the brain structures that West and Goodlett (1990) have identified as being affected by third trimester equiva lent alcohol exposure in rats.Of course, because the human data results are from correlational studies, it is possible that the observed deficits stem from other factors (e.g., the childrearing environment) rather than any specific neurological deficits that result from alcohol exposure.

CONCLUSIONS
Despite the extensive research on effects of prenatal alcohol exposure, information about timing of exposure as it relates to the fetus' vulnerability to particular ef fects on behaviors remains limited.Due to the nature of the developmental proc ess, there are differences in the amount of knowledge available about specific effects of all kinds.Animal studies and epidemiologic studies strongly suggest that the facial malformation characteristic of FAS results from exposure during the first trimester and, more specifically, during the first 2 months of gestation.However, the relationship becomes less clear in the examination of growth retardation.It appears that both early exposure (during the first 2 months of pregnancy) and exposure during the third trimester affect growth.Specifically, ef fects on head circumference-and, by extension, brain growth-appear to be the most consistent and permanent outcomes of exposure during these two periods.
It is much more difficult to examine behavioral effects than physical anomalies, using a trimester approach, due to the nature of behavior development.Correlational studies suggest that early exposure may be more damaging to behavior than is later exposure, but some animal studies indicate that exposure in the third trimester may specifically affect the hippocampus and the cerebellum, leading to deficits in learning and motor skills.However, the many social and physical factors associated with heavy alcohol use by pregnant women as well as the difficulty in studying specific behavioral effects in children limit the interpretations.The available evidence strongly suggests that there may be specific behavioral effects of alcohol exposure during particular peri ods, but the limitations inherent in both animal and human studies make it difficult to be sure of these outcomes.
It also is clear from these data that although a mother's heavy alcohol use in pregnancy is potentially damaging to the fetus, her stopping use is likely to have a beneficial outcome even on many of the functions (e.g., growth and behavior) that were affected by earlier drinking.In the future, animal studies may illuminate spe cific brain structures and patterns of behav ior that are impacted by certain patterns of alcohol exposure (West and Goodlett 1990).It may be possible to relate such findings to similar brain areas and behavior patterns in humans and thereby greatly increase under standing of these problems.■

Figure 2
Figure 2 Timing of the brain growth spurt during development in humans and rats.In the rat (scale is measured in days), the brain's growth peaks after birth, whereas in humans (scale is measured in months), the brain's growth peaks at birth.Growth is measured as a percentage of adult weight.As brain growth slows, continued growth in the rest of the body causes the brain's weight to become a smaller percentage of total adult weight.

Figure 4
Figure 4 Reevaluation of growth in alcohol-exposed children at 5 to 7 years of age suggests that effects on weight and height are mitigated, whereas effects on head circumference remain.Children whose mothers drank throughout pregnancy show the greatest deficits in head circumference.SOURCE: Coles et al. 1991.